1. Field of the Invention
The invention in the fields of microbiology and medicine relates to methods for rapid early detection of mycobacterial disease in humans, particularly tuberculosis (TB) based on the presence of antibodies to particular “early” mycobacterial antigens in the form of peptide epitopes which have not been previously recognized for this purpose. Assay for such antibodies using these early peptide epitopes permits diagnosis of TB earlier than has been heretofore possible. The invention is also directed to vaccine compositions and methods useful for preventing or treating TB by immunizing with such peptide-based antigens.
2. Description of the Background Art
Globally, TB kills ˜3 million and infects ˜9 million new individuals every year. The HIV-epidemic has exacerbated the TB epidemic in TB-endemic countries and has led to TB becoming the leading cause of morbidity and mortality in this highly vulnerable population. The development of new drugs, vaccines and diagnostic tests for TB is a major priority and towards this goal, the genomes of Mycobacterium tuberculosis (Mtb) H37Rv, and subsequently several clinical isolates of Mtb, and some non-tuberculous pathogenic and non-pathogenic mycobacteria have been, or are being currently sequenced (7, 13, 23, 52) (See also, the WWW URL ncbi.nlm.nih.gov/sutils/genom_table.cgi). The availability of these resources has accelerated the pace of research aimed at devising rational strategies for TB control.
It is well established that Mtb adapts to the changing environmental conditions during the course of progression of infection to clinical disease by differential gene expression (6, 38, 41, 48, 49, 53, 54). In the quest to understand the host-pathogen interactions that lead to establishment of Mtb infection, the present inventors and colleagues had used sera from Mtb aerosol-infected rabbits for immuno-screening a λgt11 expression library of Mtb genomic DNA (49) and published patent applications. These sera identified several Mtb proteins that contain tandem repeats of unique amino acid motifs in their sequence, and were either known or predicted to be surface/secreted proteins (49).
One of these repetitive proteins was a Proline-Threonine Repetitive Protein (PTRP; Rv0538) that is annotated as a hypothetical cell-membrane protein and classified to the functional category of cell-wall and cell processes in the Mtb H37Rv genome (7, 49). Although there is no sequence similarity, the domain organization of PTRP is reminiscent of the heparin-binding hemagglutinin (HBHA; Rv0475) of Mtb and the laminin-binding protein (ML-LBP21; ML1683) of M. leprae, in that all three proteins have repeats of specific amino-acid motifs clustered towards the C-terminus (29, 46, 49).
Another Mtb cell wall protein, PE-PGRS51 (Rv3367) has multiple tandem repeats of unique amino-acid sequences, and characteristics of surface or secreted proteins.
A third protein, LipC (originally identified as Rv0220) is a 403 amino acid, 44 kDa protein, annotated as a probable esterase in the Mtb database based on a putative carboxylesterases type-B serine active site. It is a member of a family of 24 proteins, two of which (LipY and LipH) have been shown to be induced during starvation and under acidic conditions.
There is a need in the art to identify constituents of Mtb, primarily proteins, or fragments thereof with B cell epitopes that can serve as (a) antigens in immunoassays for early detection of mycobacterial disease and/or (b) a basis for immunogens or vaccines to induce anti-mycobacterial antibody responses that are prophylactic or therapeutic. The present invention is directed to three such proteins and particular immunodominant peptide fragments thereof.
No proteomic studies of Mtb culture filtrates, cytosol, cell-wall or membrane fraction have identified PTRP although ptrp transcripts are reported to be downregulated in Mtb sigma factor sigF mutant strain compared to wild type Mtb in broth culture, suggesting that ptrp is expressed during growth in broth culture and its expression is regulated by sigF (2, 15, 17, 20, 25-27, 40, 44, 50, 51, 55). Similarly, there is no information available as to the expression of PE-PGRS51 or LipC as above. The present inventors have identified these proteins and specific peptides thereof as useful antigens for early detection or immunization.
Citation of the above documents is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicant and does not constitute any admission as to the correctness of the dates or contents of these documents.